The present invention relates to a dermal scaffold and a bioartificial dermis using the same. More specifically, the present invention relates to the dermal scaffold and the bioartificial dermis comprising alkaline pre-treated free amine-containing chitosan matrix, alkaline pre-treated free amine-containing chitosan and alkaline pre-treated collagen mixed matrix, or alkaline pre-treated free amine-containing chitosan and alkaline pre-treated collagen mixed matrix containing chitosan fabrics, which are extremely useful for wound healing therapy.
Various kinds of ordinary dressings for wound healing have been hitherto developed and conveniently used, but in most cases, they have been used only for the prevention of infection or dehydration.
Recently, acellular artificial skins or cell-based bioartificial skins have been developed and marketed by many biotechnological companies. As examples, acellular artificial skins, such as an acellular collagen-glycosaminoglycan matrix bonded to a thin silicone membrane (INTEGRA(trademark), Interga LifeSciences Co.) and dehydrorothermally cross-linked composites of fibrillar and denatured collagens (Terudermis(trademark), Terumo Co.), are now commercially available. However, such products are very expensive because they incorporate biomaterials such as collagen and thus, have difficulty in clinical trials on broad wound sites, e.g., burns.
As cell-based bioartificial skins, Advanced Tissue Sciences, Inc. (La Jolla, Calif.) developed a skin replacement product composed of a thin biodegradable mesh framework onto which human dermal fibroblasts (hereinafter, abbreviated as xe2x80x9cHDFxe2x80x9d on occasion) are seeded, for use in treating diabetic foot ulcers (Dermagraft-TC(trademark)). In addition, epidermal cell sheet for partial-thickness wound (Acticel(trademark), Biosurface Technology, Inc.), composite grafts of cultured keratinocytes and fibroblasts on a collagen glycosaminoglycan matrix (Apligraft(trademark), Organogenesis, Inc.) and a skin replacement product derived from human cadaver skin (Alloderm(trademark), Lifecell), etc., were developed.
The cell-based bioartificial skins were prepared by primary culture of human dermal fibroblasts and keratinocytes followed by 3-dimensional culture (3-D culture, raft culture) of the cultured cells in hydrated collagen. They had considerably good wound healing and scar reducing effects in clinical trials on bum or plastic surgery patients. However, they still have problems in that they are too expensive due to incorporation of collagen (e.g. Dermagraft-TC: $2,000/10xc3x9710 cm, Terudermis: $1,500) and are limited in their uses as grafts due to a low rigidity of hydrated collagen gel. Therefore, there still exists an important demand for development of polymers with good biocompatibility and biodegradability, which can successfully replace collagen and is also suitable for use as a scaffold.
To be a dermal scaffold, porous microstructures are required either to allow tissue ingrowth in vivo or to provide a template for directed tissue assembly in vitro. The skin equivalent is ideally reconstructed by grafting human epidermal keratinocytes onto a porous non-contractile dermal equivalent populated with mitotically and metabolically active HDFs. Among natural polymers that can be easily formed into a porous spongy matrix, there is a particular interest in chitosan. Chitosan is a linear polysaccharide obtained from partial deacetylation of chitin that can be derived from arthropod exoskeletons. Chitin is slowly degraded in vivo and thus, chitin and its degradation products are natural and safe. In the pharmaceutical field, chitosan has been used as a vehicle for the sustained release of drugs (Hou et al., Chem Pharm Bull 1985; 33(9):3986-3992). Chitin as such has been woven into fabrics and used as dressings for wound healing.
Particularly, Lorenz et al. (U.S. Pat. No. 5,420,197) described a hydrophilic hydrogel which comprised a blend of acid neutralized water-soluble chitosan and poly(N-vinyl lactam). In the patent, neutralized water-soluble chitosan means chitosan protonated with acids, which becomes soluble in water, e.g. salts with pyrrolidone carboxylic acid, glutamic acid, acetic acid, etc. and N,O-carboxymethyl chitosan (NOCC). The resulting gel may be used as a wound dressing because of its non-adherence property to the wound.
Berscht et al. (Biomaterials 1994; 15(8); 593-600) disclosed methylpyrrolidinone chitosan (MPC), one of the water-soluble chitosan derivatives, as a carrier material for growth factors. However, water-soluble chitosan derivatives are easily dissolved in water, PBS, saline or culture media. They cannot maintain the original shapes and the structural integrity for the ingrowth of stromal fibroblasts and the formation of microvessel, which is an essential feature of a dermal scaffold.
Hansbrough et al. (U.S. Pat. No. 5,460,939) described a temporary living skin replacement comprising: a) a living stromal tissue enveloping b) a three-dimensional structural framework composed of a biodegradable or non-biodegradable material, and c) a transitional covering made of silicone or polyurethane. According to the patent, the framework may be composed of chitosan or its derivatives. However, it is a mesh type, which is more brittle and stiff, and less tensile than a spongy type. Further, NOCC, one example of a chitosan derivative described, is soluble in water. NOCC can be spun into fiber using coagulation baths containing Ca2+ or other similar di- or trivalent cations. However, the detached cations from degraded dressing could cause significant damages to stromal cells.
Therefore, it has never been reported that chitosan matrix having free amine group by alkaline pre-treatment of an acidic chitosan solution, which is insoluble in water, can be used as a dermal scaffold, providing a structural integrity for migration and proliferation of fibroblasts and vascular endothelial cells surrounding wound site.
Accordingly, in order to solve the above-mentioned problems involved in the prior art, an object of the present invention is to provide a novel dermal scaffold for wound healing with excellent biocompatibility and biodegradability, which is not only cost-effective but also convenient to manipulate owing to its improved rigidity. In one aspect of the invention, the dermal scaffold further comprises one or more growth factors, e.g., such as basic fibroblast growth factor (bFGF), fibronectin, etc., for accelerating wound healing.
Another object of the present invention is to provide a bioartificial dermis wherein human fibroblasts are loaded onto the dermal scaffold, particularly useful for healing of broad wound sites such as burns.
In order to attain the above-described objects, one aspect of the present invention provides a dermal scaffold comprising alkaline pre-treated chitosan matrix, alkaline pre-treated chitosan and alkaline pre-treated collagen mixed matrix, or alkaline pre-treated chitosan and alkaline pre-treated collagen mixed matrix containing chitosan fabrics. The dermal scaffold preferably further comprises one or more selected from the group consisting of fibronectin, basic fibroblast growth factor, epidermal growth factor and transforming growth factor-xcex2.
Another aspect of the present invention provides a bioartificial dermis wherein human fibroblasts are loaded onto alkaline pre-treated chitosan matrix, alkaline pre-treated chitosan and alkaline pre-treated collagen mixed matrix, or alkaline pre-treated chitosan and alkaline pre-treated collagen mixed matrix containing chitosan fabrics.
In the present invention, chitosan is pre-treated with an alkaline solution thereby to become insoluble in water. The matrices of the present invention are characterized in that they are made of such the alkaline pre-treated chitosan that is insoluble in an aqueous medium or a body fluid. Therefore, it can provide a structural integrity for migration and proliferation of wound healing cells, such as fibroblasts and vascular endothelial cells, surrounding wound site.